Ionic Charge and pH Regulation of Apoptotic Signaling

细胞凋亡信号传导的离子电荷和 pH 调节

• 批准号: 6360323
• 负责人: MARK S. SEGAL
• 金额: $ 7.25万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6360323
• 关键词: acid base balance; animal tissue; apoptosis; biological signal transduction; cysteine endopeptidases; cytochrome c; tissue /cell culture; vascular endothelium

This proposal is in response to PAR-98-087, "Small grant program for K08 recipients," and is linked to NIH K08 DK02537. Endothelial cell apoptosis has an essential role in the pathogenesis of hemolytic uremic syndrome and other causes of acute renal failure. The studies in this proposal will advance our understanding of the biochemistry of endothelial cell apoptosis by examining the molecular mechanism by which ionic charge and alkaline conditions inhibit cytochrome c- mediated activation of caspase-3. We have developed an in vitro assay of cytochrome c-dependent activation of cytosolic caspase-3 allowing discrete steps in the process to be identified and characterized. In this assay cytochrome c added to cytosol initiates the formation of apoptosomes (oligomers of cytochrome c, apoptosis activating factor-1, and dATP/ATP) resulting in activation of caspase-9, which subsequently activates caspase-3. We showed that both alkaline pH and cations, at normal intracellular concentrations, inhibited activation of caspase-3. Excess cytochrome c suppressed the inhibition by high pH, but did not affect the inhibition by cations, suggesting that pH and cationic strength act at two different steps in caspase-3 activation. This proposal will test two overall hypotheses derived from these observations. First, that alkaline pH affects the formation of apoptosomes, whereas ionic charge prevents caspase-9 activation. Second, that cellular accumulation of a non-cationic osmotic solute is required for the decrease in cationic strength necessary for caspase-3 activation. The Specific Aims are 1) to develop an in vitro assay for caspase-9 activation, and determine the effect of alkalinity and high cationic strength on activation; 2) to study the formation and constituents of apoptosomes, using a size separation column, under high ionic charge and alkaline conditions; and 3) to examine the role of non- cationic solute accumulation in apoptosis by testing if potential osmoles, such as amino acids and mono- and di-saccharides, interfere with caspase-3 activation in our in vitro assay, and by comparing cytosol from nonapoptotic and apoptotic cells using liquid chromatography for differences in osmotic solute content. This grant will also enhance the research productivity, fiscal independence and career development of the applicant furthering his advancement towards becoming an independent investigator.

该提案是对98-087 PAR-98-087的回应，即“ K08接收者的小型赠款计划”，并与NIH K08 DK02537相关。内皮细胞凋亡在溶血性尿毒症综合征的发病机理和其他导致急性肾衰竭的原因中具有至关重要的作用。该提案中的研究将通过检查分子机制，通过研究离子电荷和碱性条件抑制细胞色素c-介导的caspase-3激活的分子机制，以提高我们对内皮细胞凋亡的生物化学的理解。我们已经开发了细胞色素C依赖性活化的胞质caspase-3的体外测定，从而可以在过程中识别和表征离散的步骤。在此测定中，添加到细胞质中的细胞色素C启动了凋亡体（细胞色素C的低聚物，凋亡激活因子1和DATP/ATP）的形成，从而激活Caspase-9，随后激活Caspase-3。我们表明，在正常细胞内浓度下，碱性pH和阳离子都抑制了caspase-3的激活。多余的细胞色素C抑制了高pH值的抑制作用，但不会影响阳离子的抑制作用，这表明pH和阳离子强度在caspase-3激活中以两个不同的步骤作用。该提案将检验从这些观察结果中得出的两个总体假设。首先，碱性pH会影响凋亡组的形成，而离子电荷则阻止了caspase-9激活。其次，对于caspase-3激活所需的阳离子强度降低，需要非阳离子渗透溶质的细胞积累。具体目的是1）开发用于caspase-9激活的体外测定，并确定碱度和高阳离子强度对激活的影响； 2）在高离子电荷和碱性条件下，使用尺寸分离柱研究凋亡小体的形成和成分； 3）通过测试潜在的渗透量（例如氨基酸和单糖和二糖），研究非阳离子溶质积累在凋亡中的作用，从而在我们的体外测定中干扰了Caspase-3激活，并通过使用液体色谱物中的液体色谱溶液中的溶液中的溶液中的液体溶液中的液体化合物比较我们的体外测定中的活化。这项赠款还将提高申请人的研究生产率，财政独立性和职业发展，从而进步成为独立研究者。